(Hydroxyphenyl)-1H-Indole-3-Carbaldehyde Oxime Derivatives As Estrogenic Agents

ABSTRACT

This invention provides estrogen receptor modulators of formula I, having the structure  
                 
 
where R 1 , R 2 , R 3 , and R 4  are as defined in the specification, or a pharmaceutically acceptable salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/914,747, filed Aug. 9, 2004 and claims benefit to U.S.Provisional Application Ser. No. 60/494,365, filed Aug. 12, 2003, eachof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to (hydroxyphenyl)-1H-indole-3-carbaldehyde oximederivatives, their uses as estrogenic agents, and methods of theirpreparation.

BACKGROUND OF THE INVENTION

The pleiotropic effects of estrogens in mammalian tissues have been welldocumented, and it is now appreciated that estrogens affect many organsystems [Mendelsohn and Karas, New England Journal of Medicine 340:1801-1811 (1999), Epperson, et al., Psychosomatic Medicine 61: 676-697(1999), Crandall, Journal of Womens Health & Gender Based Medicine 8:1155-1166 (1999), Monk and Brodaty, Dementia & Geriatric CognitiveDisorders 11:1 -10 (2000), Hum and Macrae, Journal of Cerebral BloodFlow & Metabolism 20: 631-652 (2000), Calvin, Maturitas 34: 195-210(2000), Finking, et al., Zeitschrift fur Kardiologie 89: 442-453 (2000),Brincat, Maturitas 35: 107-117 (2000), Al-Azzawi, Postgraduate MedicalJournal 77: 292-304 (2001)]. Estrogens can exert effects on tissues inseveral ways. Probably, the most well characterized mechanism of actionis their interaction with estrogen receptors leading to alterations ingene transcription. Estrogen receptors are ligand-activatedtranscription factors and belong to the nuclear hormone receptorsuperfamily. Other members of this family include the progesterone,androgen, glucocorticoid and mineralocorticoid receptors. Upon bindingligand, these receptors dimerize and can activate gene transcriptioneither by directly binding to specific sequences on DNA (known asresponse elements) or by interacting with other transcription factors(such as AP1), which in turn bind directly to specific DNA sequences[Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall, et al.,Journal of Biological Chemistry 276: 36869-36872 (2001), McDonnell,Principles Of Molecular Regulation. p351-361(2000)]. A class of“coregulatory” proteins can also interact with the ligand-bound receptorand further modulate its transcriptional activity [McKenna, et al.,Endocrine Reviews 20: 321-344 (1999)]. It has also been shown thatestrogen receptors can suppress NFκB-mediated transcription in both aligand-dependent and independent manner [Quaedackers, et al.,Endocrinology 142: 1156-1166 (2001), Bhat, et al., Journal of SteroidBiochemistry & Molecular Biology 67: 233-240 (1998), Pelzer, et al.,Biochemical & Biophysical Research Communications 286: 1153-7 (2001)].

Estrogen receptors can also be activated by phosphorylation. Thisphosphorylation is mediated by growth factors such as EGF and causeschanges in gene transcription in the absence of ligand [Moggs andOrphanides, EMBO Reports 2: 775-781 (2001), Hall, et al., Journal ofBiological Chemistry 276: 36869-36872 (2001)].

A less well-characterized means by which estrogens can affect cells isthrough a so-called membrane receptor. The existence of such a receptoris controversial, but it has been well documented that estrogens canelicit very rapid non-genomic responses from cells. The molecular entityresponsible for transducing these effects has not been definitivelyisolated, but there is evidence to suggest it is at least related to thenuclear forms of the estrogen receptors [Levin, Journal of AppliedPhysiology 91: 1860-1867 (2001), Levin, Trends in Endocrinology &Metabolism 10: 374-377 (1999)].

Two estrogen receptors have been discovered to date. The first estrogenreceptor was cloned about 15 years ago and is now referred to as ERα[Green, et al., Nature 320: 134-9 (1986)]. The second was foundcomparatively recently and is called ERβ [Kuiper, et al., Proceedings ofthe National Academy of Sciences of the United States of America 93:5925-5930 (1996)]. Early work on ERβ focused on defining its affinityfor a variety of ligands and, indeed, some differences with ERα wereseen. The tissue distribution of ERβ has been well mapped in the rodentand it is not coincident with ERα. Tissues such as the mouse and ratuterus express predominantly ERα, whereas the mouse and rat lung expresspredominantly ERβ [Couse, et al., Endocrinology 138: 4613-4621 (1997),Kuiper, et al., Endocrinology 138: 863-870 (1997)]. Even within the sameorgan, the distribution of ERα and ERβ can be compartmentalized. Forexample, in the mouse ovary, ERβ is highly expressed in the granulosacells and ERα is restricted to the thecal and stromal cells [Sar andWelsch, Endocrinology 140: 963-971 (1999), Fitzpatrick, et al.,Endocrinology 140: 2581-2591 (1999)]. However, there are examples wherethe receptors are coexpressed and there is evidence from in vitrostudies that ERα and ERβ can form heterodimers [Cowley, et al., Journalof Biological Chemistry 272: 19858-19862 (1997)].

The most potent endogenous estrogen is 17β-estradiol. A large number ofcompounds have been described that either mimic or block the activity of17β-estradiol. Compounds having roughly the same biological effects as17β-estradiol are referred to as “estrogen receptor agonists”. Thosewhich block the effects of 17β-estradiol, when given in combination withit, are called “estrogen receptor antagonists”. In reality, there is acontinuum between estrogen receptor agonist and estrogen receptorantagonist activity and some compounds behave as estrogen receptoragonists in some tissues but estrogen receptor antagonists in others.These compounds with mixed activity are called selective estrogenreceptor modulators (SERMS) and are therapeutically useful agents (e.g.EVISTA) [McDonnell, Journal of the Society for Gynecologic Investigation7: S10-S15 (2000), Goldstein, et al., Human Reproduction Update 6:212-224 (2000)]. The precise reason why the same compound can havecell-specific effects has not been elucidated, but the differences inreceptor conformation and/or in the milieu of coregulatory proteins havebeen suggested.

It has been known for some time that estrogen receptors adopt differentconformations when binding ligands. However, the consequence andsubtlety of these changes only recently has been revealed. The threedimensional structures of ERα and ERβ have been solved byco-crystallization with various ligands and clearly show therepositioning of helix 12 in the presence of an estrogen receptorantagonist, which sterically hinders the protein sequences required forreceptor-coregulatory protein interaction [Pike, et al., Embo 18:4608-4618 (1999), Shiau, et al., Cell 95: 927-937 (1998)]. In addition,the technique of phage display has been used to identify peptides thatinteract with estrogen receptors in the presence of different ligands[Paige, et al., Proceedings of the National Academy of Sciences of theUnited States of America 96: 3999-4004 (1999)]. For example, a peptidewas identified that distinguished between ERα bound to the full estrogenreceptor agonists 17β-estradiol and diethylstilbesterol. A differentpeptide was shown to distinguish between clomiphene bound to ERα andERβ. These data indicate that each ligand potentially places thereceptor in a unique and unpredictable conformation that is likely tohave distinct biological activities.

As mentioned above, estrogens affect a panoply of biological processes.In addition, where gender differences have been described (e.g. diseasefrequencies, responses to challenge, etc), it is possible that theexplanation involves the difference in estrogen levels between males andfemales.

SUMMARY OF THE INVENTION

This invention relates to (hydroxyphenyl)-1H-indole-3-carbaldehyde oximederivatives. In certain aspects, the invention relates to compounds offormula I:

wherein:

-   -   R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy;    -   R₂ is hydrogen, lower alkyl, or phenyl;    -   or R₁ and R₂ together may form a 5-7 membered ring; and    -   R₃, and R₄ are each, independently, H, OH, halogen, CN, phenyl,        substituted phenyl, lower alkyl or lower alkoxy; or a        pharmaceutically acceptable salt or prodrug thereof.

In certain embodiments, the invention concerns a pharmaceuticalcomposition comprising compound 1 and a pharmaceutically acceptablecarrier.

In other embodiments, the invention is directed to the use of suchcompounds in the treatment or prevention of diseases such asinflammatory bowel diseases.

DETAILED DESCRIPTIION OF THE INVENTION

This invention provides estrogenic compounds of formula I, which isuseful in the treatment and/or prevention of diseases such asinflammatory bowel diseases (including Crohn's disease and colitis).

where R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂ ishydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a 5-7membered ring; and R₃, and R₄ are each, independently, H, OH, halogen,CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; or apharmaceutically acceptable salt or prodrug thereof.

In certain embodiments of compound 1, R₁ is lower alkyl. In otherembodiments, R₂ is hydrogen, or alkyl. Further embodiments encompasscompositions where R₁ and R₂ together form a 6 membered ring. In someembodiments, R₃ is H, halogen or CN. R₄ is hydrogen in some embodiments.In some other embodiments, R₄ is fluoro. In some embodiments, R4 isortho fluoro.

Pharmaceutically acceptable salts can be formed from organic andinorganic acids, for example, acetic, propionic, lactic, citric,tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,camphorsulfonic, and similarly known acceptable aids when a compound ofthis invention contains a basic moiety. Salts may also be formed fromorganic and inorganic bases, such as alkali metal salts (for example,sodium, lithium, or potassium) alkaline earth metal salts, ammoniumsalts, alkylammonium salts containing 1-6 carbon atoms ordialkylammonium salts containing 1-6 carbon atoms in each alkyl group,and trialkylammonium salts containing 1-6 carbon atoms in each alkylgroup, when a compound of this invention contains an acidic moiety.

The term “alkyl”, as used herein, whether used alone or as part ofanother group, refers to a substituted or unsubstituted aliphatichydrocarbon chain and includes, but is not limited to, straight andbranched chains containing from 1 to 12 carbon atoms, preferably 1 to 6carbon atoms, unless explicitly specified otherwise. For example,methyl, ethyl, propyl, isopropyl, butyl, i-butyl and t-butyl areencompassed by the term “alkyl.” Specifically included within thedefinition of “alkyl” are those aliphatic hydrocarbon chains that areoptionally substituted.

The term “lower alkyl” refers to an alkyl group having 1 to 6 carbonatoms, in some embodiments 1 to 3 carbon atoms.

The carbon number as used in the definitions herein refers to carbonbackbone and carbon branching, but does not include carbon atoms of thesubstituents, such as alkoxy substitutions and the like.

The term “alkoxy,” as used herein, refers to the group R—O— where R isan alkyl group preferably of 1 to 6 carbon atoms.

The term “alkenyl”, as used herein, whether used alone or as part ofanother group, refers to a substituted or unsubstituted aliphatichydrocarbon chain and includes, but is not limited to, straight andbranched chains having 2 to 8 carbon atoms and containing at least onedouble bond. Preferably, the alkenyl moiety has 1 or 2 double bonds.Such alkenyl moieties may exist in the E or Z conformations and thecompounds of this invention include both conformations. Specificallyincluded within the definition of “alkenyl” are those aliphatichydrocarbon chains that are optionally substituted. Heteroatoms, such as0, S or N—R₁, attached to an alkenyl should not be attached to a carbonatom that is bonded to a double bond.

When used herein the term alkynyl refers to an aliphatic, straight orbranched, hydrocarbon chain having 2 to 7 carbon atoms that may contain1 to 3 triple bonds. When used herein, the term cycloalkyl refers to amonocarbocyclic ring having 3 to 8 carbon atoms e.g. cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl. When used herein, alone or aspart of a group, the term aryl refers to an aromatic 5 to 13 memberedmono- or bi-carbocyclic ring such as phenyl or naphthyl. Preferably,groups containing aryl moieties are monocyclic having 5 to 7 carbonatoms in the ring. When used herein alone or as part of a group the termheteroaryl refers to an aromatic 5 to 13-membered carbon containing monoor bicyclic ring having one to five heteroatoms which independently maybe nitrogen, oxygen or sulfur. Preferably, groups containing heteroarylmoieties are monocyclic having 5 to 7 members in the ring where one ortwo of the ring members are independently selected from nitrogen, oxygenor sulfur. Suitable aryl groups include furanyl, thienyl, pyridinyl,indolyl and quinolyl.

Examples for a halogen substituented alkyls and alkenyls include 1-bromovinyl, 1-fluoro vinyl, 1,2-difluoro vinyl, 2,2-difluorovinyl,1,2,2-trifluorovinyl, 1,2-dibromo ethane, 1,2 difluoro ethane,1-fluoro-2-bromo ethane, CF₂CF₃, CF₂CF₂CF₃, and the like.

The term “phenyl”, as used herein, whether used alone or as part ofanother group, refers to a substituted or unsubstituted phenyl group.

An optionally substituted alkyl, alkenyl, and phenyl may be substitutedwith one or more substituents. Suitable optionally substituents may beselected independently from nitro, cyano, —N(R₁₁)(R₁₂), halo, hydroxy,carboxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylalkoxy, al koxycarbonyl, alkoxyalkoxy,perfluoroalkyl, perfluoroalkoxy, arylalkyl, alkylaryl, hydroxyalkyl,alkoxyalkyl, alkylthio, —S(O)₂—N(R₁₁)(R₁₂), —C(═O)—N(R₁₁)(R₁₂),(R₁₁)(R₁₂)N-alkyl, (R₁₁)(R₁₂)N-alkoxyalkyl,(R₁₁)(R₁₂)N-alkylaryloxyalkyl, —S(O)_(s)-aryl (where s=0−2), or—S(O)_(s)-heteroaryl (where s=0−2). In certain embodiments of theinvention, preferred substituents for alkyl, alkenyl, alkynyl, phenyland cycloalkyl include nitro, cyano, —N(R₁₁)(R₁₂), halo, hydroxyl, aryl,heteroaryl, alkoxy, alkoxyalkyl, and alkoxycarbonyl. In certainembodiments of the invention, preferred substituents for aryl andheteroaryl include —N(R₁₁)(R₁₂), alkyl, halo, perfluoroalkyl,perfluoroalkoxy, arylalkyl and alkylaryl. R₁₁ and R₁₂ are eachindependently selected from hydrogen and alkyl.

The term “lower alkoxy,” as used herein, refers to the group R—O— whereR is an alkyl group of 1 to 6 carbon atoms.

The term halogen includes bromine, chlorine, fluorine, and iodine.

As used in accordance with this invention, the term “providing,” withrespect to providing a compound or substance covered by this invention,means either directly administering such a compound or substance, oradministering a prodrug, derivative, or analog which will form theeffective amount of the compound or substance within the body.

The reagents used in the preparation of the compounds of this inventioncan be either commercially obtained or can be prepared by standardprocedures described in the literature.

The preparation of several representative examples of this invention aredescribed in the following Schemes 1-3.

The compounds of this invention are estrogen receptor modulators usefulin the treatment or inhibition of conditions, disorders, or diseasestates that are at least partially mediated by an estrogen deficiency orexcess, or which may be treated or inhibited through the use of anestrogenic agent. The compounds of this invention are particularlyuseful in treating a peri-menopausal, menopausal, or postmenopausalpatient in which the levels of endogenous estrogens produced are greatlydiminished. Menopause is generally defined as the last natural menstrualperiod and is characterized by the cessation of ovarian function,leading to the substantial diminution of circulating estrogen in thebloodstream. As used herein, menopause also includes conditions ofdecreased estrogen production that may be surgically, chemically, or becaused by a disease state which leads to premature diminution orcessation of ovarian function.

Accordingly, the compounds of this invention are useful in treating orinhibiting osteoporosis and in the inhibition of bone demineralization,which may result from an imbalance in a individual's formation of newbone tissues and the resorption of older tissues, leading to a net lossof bone. Such bone depletion results in a range of individuals,particularly in post-menopausal women, women who have undergonebilateral oophorectomy, those receiving or who have received extendedcorticosteroid therapies, those experiencing gonadal dysgenesis, andthose suffering from Cushing's syndrome. Special needs for bone,including teeth and oral bone, replacement can also be addressed usingthese compounds in individuals with bone fractures, defective bonestructures, and those receiving bone-related surgeries and/or theimplantation of prosthesis. In addition to those problems describedabove, these compounds can be used in treatment or inhibition forosteoarthritis, hypocalcemia, hypercalcemia, Paget's disease,osteomalacia, osteohalisteresis, multiple myeloma and other forms ofcancer having deleterious effects on bone tissues.

The compounds of this invention are also useful in treating orinhibiting benign or malignant abnormal tissue growth, includingprostatic hypertrophy, uterine leiomyomas, breast cancer, endometriosis,endometrial cancer, polycystic ovary syndrome, endometrial polyps,benign breast disease, adenomyosis, ovarian cancer, melanoma, prostratecancer, cancers of the colon, CNS cancers, such as glioma orastioblastomia.

The compounds of this invention are cardioprotective and they are usefulin lowering cholesterol, triglycerides, Lp(a), and LDL levels;inhibiting or treating hypercholesteremia; hyperlipidemia;cardiovascular disease; atherosclerosis; peripheral vascular disease;restenosis, and vasospasm; and inhibiting vascular wall damage fromcellular events leading toward immune mediated vascular damage. Thesecardiovascular protective properties are of great importance whentreating postmenopausal patients with estrogens to inhibit osteoporosisand in the male when estrogen therapy is indicated.

The compounds of this invention are also antioxidants, and are thereforeuseful in treating or inhibiting free radical induced disease states.Specific situations in which antioxidant therapy is indicated to bewarranted are with cancers, central nervous system disorders,Alzheimer's disease, bone disease, aging, inflammatory disorders,peripheral vascular disease, rheumatoid arthritis, autoimmune diseases,respiratory distress, emphysema, prevention of reperfusion injury, viralhepatitis, chronic active hepatitis, tuberculosis, psoriasis, systemiclupus erythematosus, adult respiratory distress syndrome, centralnervous system trauma and stroke.

The compounds of this invention are also useful in providing cognitionenhancement, and in treating or inhibiting senile dementias, Alzheimer'sdisease, cognitive decline, neurodegenerative disorders, providingneuroprotection or cognition enhancement.

The compounds of this invention are also useful in treating orinhibiting inflammatory bowel disease, ulcerative proctitis, Crohn'sdisease, and colitis; menopausal related conditions, such as vasomotorsymptoms including hot flushes, vaginal or vulvar atrophy, atrophicvaginitis, vaginal dryness, pruritus, dyspareunia, dysuria, frequenturination, urinary incontinence, urinary tract infections, vasomotorsymptoms, including hot flushes, myalgia, arthralgia, insomnia,irritability, and the like; male pattern baldness; skin atrophy; acne;type II diabetes; dysfunctional uterine bleeding; and infertility.

The compounds of this invention are useful in disease states whereamenorrhea is advantageous, such as leukemia, endometrial ablations,chronic renal or hepatic disease or coagulation diseases or disorders.

The compounds of this invention can be used as a contraceptive agent,particularly when combined with a progestin.

When administered for the treatment or inhibition of a particulardisease state or disorder, it is understood that the effective dosagemay vary depending upon the particular compound utilized, the mode ofadministration, the condition, and severity thereof, of the conditionbeing treated, as well as the various physical factors related to theindividual being treated. Effective administration of the compounds ofthis invention may be given at an oral dose of from about 0.1 mg/day toabout 1,000 mg/day. Preferably, administration will be from about 10mg/day to about 600 mg/day, more preferably from about 50 mg/day toabout 600 mg/day, in a single dose or in two or more divided doses. Theprojected daily dosages are expected to vary with route ofadministration.

Such doses may be administered in any manner useful in directing theactive compounds herein to the recipient's bloodstream, includingorally, via implants, parenterally (including intravenous,intraperitoneal and subcutaneous injections), rectally, intranasally,vaginally, and transdermally.

Oral formulations containing the active compounds of this invention maycomprise any conventionally used oral forms, including tablets,capsules, buccal forms, troches, lozenges and oral liquids, suspensionsor solutions. Capsules may contain mixtures of the active compound(s)with inert fillers and/or diluents such as the pharmaceuticallyacceptable starches (e.g. corn, potato or tapioca starch), sugars,artificial sweetening agents, powdered celluloses, such as crystallineand microcrystalline celluloses, flours, gelatins, gums, etc. Usefultablet formulations may be made by conventional compression, wetgranulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. Preferred surface modifyingagents include nonionic and anionic surface modifying agents.Representative examples of surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine. Oral formulations herein mayutilize standard delay or time release formulations to alter theabsorption of the active compound(s). The oral formulation may alsoconsist of administering the active ingredient in water or a fruitjuice, containing appropriate solubilizers or emulsifiers as needed.

In some cases it may be desirable to administer the compounds directlyto the airways in the form of an aerosol.

The compounds of this invention may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxy-propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparation contain a preservative to prevent thegrowth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

For the purposes of this disclosure, transdermal administrations areunderstood to include all administrations across the surface of the bodyand the inner linings of bodily passages including epithelial andmucosal tissues. Such administrations may be carried out using thepresent compounds, or pharmaceutically acceptable salts thereof, inlotions, creams, foams, patches, suspensions, solutions, andsuppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of atransdermal patch containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin. The carrier may take any number of forms such as creams andointments, pastes, gels, and occlusive devices. The creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing the active ingredientmay also be suitable. A variety of occlusive devices may be used torelease the active ingredient into the blood stream such as asemi-permeable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

Suppository formulations may be made from traditional materials,including cocoa butter, with or without the addition of waxes to alterthe suppository's melting point, and glycerin. Water soluble suppositorybases, such as polyethylene glycols of various molecular weights, mayalso be used.

EXAMPLE 1a 1-(4-Benzyloxyphenyl)-2-methyl-1H-indole

To a mixture of 2-methylindole (3.28 g, 25 mmol), 4-benzyloxyiodobenzene (7.76 g, 25 mmol), potassium carbonate (2.65 g, 25 mmol),and N-methylpyrrolidinone (250 mL) was added copper (I) bromide (0.5 g,3.5 mmol). The stirred solution was heated to 180° C. and maintained for18 hr. The reaction mixture was cooled, poured onto ice and extractedwith ethyl acetate. After washing the organic layer with water andbrine, and drying over anhydrous MgSO₄ the solvent was removed to yielda dark liquid. Purification by silica gel chromatography (2% ethylacetate-hexanes) provided the title compound as a white solid (30%): mp95-96° C.; ¹H NMR (DMSO-d₆): δ 2.24 (3H, s), 5.19 (2H, s), 6.38 (1H, s),6.93-7.04 (3H, m), 7.20 (2H, d, J=1.9Hz), 7.23 (2H, d, J=3.1 Hz),7.32-7.53 (8H, m); MS m/z (M+H)⁺314:

Anal. for C₂₂H₁₉NO 0.1H₂O:

Calc'd: C, 83.83; H, 6.14; N, 4.44.

Found: C, 83.84; H, 6.18; N, 4.29.

EXAMPLE 1b 5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole

5-Fluoro-2-methylindole (5.0 g, 33.5 mmol) and 4-benzyloxy iodobenzene(10.40 g, 33.5 mmol) were reacted according to the procedure used inExample 1a above to afford a white solid (23%): mp 94-95° C.; ¹H NMR(DMSO-d₆): δ 2.23 (3H, s), 5.19 (2H, s), 6.39 (1H, s), 6.84-6.94 (2H,m), 7.19-7.29 (3H, m), 7.34-7.44 (5H, m), 7.51 (2H, d,J=7.1 Hz); MS m/z(M+H)⁺332.

Anal. for C₂₂H₁₈NOF:

Calc'd: C, 79.74; H, 5.47; N, 4.23.

Found: C, 79.54; H, 5.54; N, 4.21.

EXAMPLE 1c 5-Chloro-1-(4-methoxymethyloxyphenyl)-2-methyl-1H-indole

5-Chloro-2-methylindole (4.14 g, 25 mmol) and 4-methoxymethyloxyiodobenzene (6.20 g, 25 mmol) were reacted according to the procedureused in Example 1a above to afford a colorless liquid (19%): ¹H NMR(DMSO-d₆): δ 2.25 (3H, s), 3.44 (2H, s), 5.28 (2H, s), 6.40 (1H, s),6.96 (1H, d, J=8.8 Hz), 7.02 (1H, dd, J=2.0 Hz, J=8.8 Hz), 7.22 (2H, d,J=8.8Hz), 7.36 (2H, d, J=8.8 Hz), 7.55 (1H, d, J=2.0 Hz); MS m/z(M+H)⁺302/304 (1 Cl).

Anal. for C₁₇H₁₆CINO₂:

Calc'd: C, 67.66; H, 5.34; N, 4.64.

Found: C, 67.49; H, 5.17; N, 4.51.

EXAMPLE 1d 5-Bromo-1-(4-methoxymethyloxyphenyl)-2-methyl-1H-indole

5-Bromo-2-methylindole (5.59 g, 23.8 mmol) and 4-methoxymethyloxyiodobenzene (7.47 g, 23.8 mmol), were reacted according to the procedureused in Example 1a above to afford a colorless liquid (8.4%): ¹H NMR(DMSO-d₆): δ 2.23 (3H, s), 3.43 (2H, s), 5.26 (2H, s), 6.39 (1H, s),6.94 (1H, d, J=8.7 Hz), 7.01 (1H, dd, J=2.2 Hz, J=8.6 Hz), 7.20 (2H, d,J=8.6H 7.34 (2H, d, J=8.6 Hz), 7.53 (1H, d, J=2.2 Hz); MS m/z(M+H)+346/348 (1 Br).

Anal. for C₁₇H₁₆BrNO₂:

Calc'd: C, 58.98; H, 4.66; N, 4.05.

Found: C, 58.72; H, 4.53; N, 4.17.

EXAMPLE 1e (4E)-9-(4-Benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazole

Carbazole (8.70 g, 39.7 mmol) and 4-benzyloxy iodobenzene (12.33 g, 39.7mmol) were reacted according to the procedure used in Example 1a aboveto afford a tan solid (20%): mp 128-129° C.; ¹H NMR (DMSO-d₆): δ 1.86(4H, br s), 2.55 (2H, br s), 2.72 (2H, br s), 5.16 (2H, s), 7.00-7.03(2H, m), 7.05-7.08 (1H, m), 7.13-7.16 (2H, m), 7.27-7.30 (2H, m),7.33-7.36 (1H, m), 7.40-7.43 (2H, m), 7.49 (2H, d, J=7.2 Hz); MS m/z(M+H)⁺354.

Anal. calcd for C₂₅H₂₃NO:

Calc'd: C, 84.95; H, 6.56; N, 3.96.

Found: C, 84.74; H, 6.76; N, 3.91.

EXAMPLE 1f 1-(4-Benzyloxy-3-fluorophenyl)-2-methyl-1H-indole

To a mixture of 2-methylindole (6.23 g, 47.5 mmol), 4-benzyloxy-3-fluorobromobenzene (14.066 g, 50 mmol), potassium carbonate (5.83 g, 55 mmol),and N-methylpyrrolidinone (500 mL) was added copper (I) bromide (1.0 g,7 mmol). The stirred solution was heated to 180° C. and maintained for18 hr. The reaction mixture was cooled, poured onto ice and extractedwith ethyl acetate. After washing the organic layer with water andbrine, and drying over anhydrous MgSO₄ the solvent was removed to yielda dark liquid, which was purified by silica gel chromatography (2% ethylacetate-hexanes) to provide the title compound alley as a white solid(3.45 gr. 22% yd): mp 70-71° C.; ¹H NMR (DMSO-d₆): δ 2.26 (3H, s), 5.28(2H, s), 6.39 (1H, s), 6.99-7.05 (3H, m), 7.21-7.24 (2H, m), 7.36-7.54(7H, m)); MS m/z (M+H)⁺332:

Anal. for C₂₂H₁₈NOF 0.1 H₂O:

Calc'd: C, 79.31; H, 5.51; N, 4.20.

Found: C, 79.29; H, 5.39; N, 4.16

EXAMPLE 2a 1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde

Phosphorous oxychloride (3 mL) was added to anhydrous dimethylformamide(6 mL) and the mixture was stirred at room temperature for 15 min. Amixture of 1-(4-benzyloxyphenyl)-2-methyl-1H-indole (0.94 g, 3 mmol) indimethylformamide (10 mL) was added and the mixture heated to 80° C. for18 hr. The reaction mixture was poured onto ice and adjusted to a pH of7 by the addition of 2 N NaOH. The mixture was extracted with ethylacetate (2×100 mL). The organic layer washed with water and brine anddried over MgSO₄. Evaporation to provide the product as a dark solid.Purification by silica gel chromatography (10% EtOAC-hexanes) provided awhite solid (63%): mp 150-152° C.; ¹H NMR (DMSO-d₆): δ 2.54 (3H, s),5.22 (2H, s), 6.99 (1H, d, J=7.9 Hz), 7.17-7.42 (4H, m), 7.45-7.54 (7H,m), 8.17 (1H, d, J=7.5 Hz), 10.19 (1H, s); MS m/z (M+H)⁺342.

Anal. for C₂₃H₁₉NO₂:

Calc'd: C, 80.92; H, 5.61; N, 4.10.

Found: C, 80.76; H, 5.70; N, 3.93.

EXAMPLE 2b5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde

Phosphorous oxychloride (5 mL) was added to anhydrous dimethylformamide(10 mL) and the mixture allowed to stir at room temperature for 15 minwhereupon a solution of5-fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole (1.63 g, 4.9 mmol) indimethylformamide (10 mL) was added and reacted according to theprocedure used in Example 2a to afford a tan solid (85%): mp 219-220°C.; ¹H NMR (DMSO-d₆): δ 2.53 (3H, s), 5.22 (2H, s), 6.99-7.06 (2H, m),7.27 (2H, d, J=8.9 Hz), 7.37-7.53 (7H, m), 7.86 (1H, dd, J=2.5 Hz, J=9.5Hz); MS m/z (M+H)⁺360.

Anal. for C₂₃H₁₈FNO₂.0.5 H₂O:

Calc'd: C, 74.99; H, 5.20; N, 3.80.

Found: C, 74.91; H, 4.96; N, 3.59.

EXAMPLE 2c5-Chloro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde

Phosphorous oxychloride (5 mL) was added to anhydrous dimethylformamide(10 mL) and stirred at room temperature for 15 min whereupon a solutionof 5-chloro-1-(4-methoxymethyloxyphenyl)-2-methyl-1H -indole (1.22 g,3.5 mmol) in dimethylformamide (10 mL) was added. The solution wasreacted according to the procedure used in Example 2a to afford a tansolid (85%): mp >240° C.; ¹H NMR (DMSO-d₆): δ 2.52 (3H, s), 6.97-7.02(3H, m), 7.22 (1H, dd, J=2.3 Hz, J=8.7 Hz), 7.30 (2H, d, J=8.6 Hz), 8.40(1H, d, J=2.1 Hz), 10.02 (1H, s), 10.16 (1H, s); MS m/Z (M+H)⁺286/288 (1Cl).

Anal. for C₁₆H₁₂CINO₂.0.3 H₂O:

Calc'd: C, 66.01; H, 4.36; N, 4.81.

Found: C, 65.85; H, 4.10; N, 4.74.

EXAMPLE 2d 5-Bromo-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde

Phosphorous oxychloride (2.5 mL) was added to anhydrousdimethylformamide (5 mL) and stirred at room temperature for 15 minwhereupon a solution of 5-bromo-1-(4-methoxymethyloxyphenyl)-2-methyl-1H-indole (0.65 g, 1.9 mmol) in dimethylformamide (10 mL) was added. Thesolution was reacted according to the procedure used in Example 2a toafford a tan solid (51%): mp >250° C.; ¹H NMR (DMSO-d₆): δ 2.50 (3H, s),6.95-7.02 (3 H, m), 7.21 (1H, dd, J=2.4 Hz, J=8.8 Hz), 7.28 (2H, d,J=8.7 Hz), 8.38 (1H, d J=2.2 Hz), 9.98 (1H, s), 10.14 (1H, s); MS m/Z(M+H)⁺318/320 (1 Br).

Anal. for C₁₆H₁₂BrNO₂.0.2 H₂0:

Calc'd: C, 55.99; H, 3.88; N, 4.35.

Found: C, 55.69; H, 3.95; N, 4.15.

EXAMPLE 2e(4E)-9-(4-Benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one

To a stirred solution of(4E)-9-(4-benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazole (1.55 g, 4.4mmol), tetrahydrofuran (30 mL), and water (20 mL) was added DDQ (1 g,4.4 mmol). Stirring was continued for 30 min whereupon the crude productwas filtered and washed with water. The product was purified by silicagel chromatography (25% EtOAc-hexanes) to give a white solid (57%): mp219-220° C.; ¹H NMR (CDCI₃): δ 2.18-2.25 (2H, m), 2.62 (2H, t, J=5.9Hz), 2.79 (2H, t, J=6.1 Hz), 5.16 (2H, s), 7.11-7.51 (12H, m), 8.30 d,J=7.9 Hz); MS m/z (M+H)⁺368.

Anal. for C₂₅H₂₁NO₂.0.5 H₂O:

Calc'd: C, 79.76; H, 5.86; N, 3.72.

Found: C, 79.86; H, 5.75; N, 3.53.

EXAMPLE 2f 1-(4-Benzyloxyphenyl)-H-indole-3-carbaldehyde

1H-Indole-3-carbaldehyde (3.63 g, 25 mmol) and 4-benzyloxy iodobenzene(7.76 g, 25 mmol) were reacted according to the procedure used inExample 1a above to afford a yellow solid (12%): mp 62-64° C.; ¹H NMR(DMSO-d₆): δ 5.22 (2H, s), 7.26 (2H, d, J=8.8 Hz), 7.32-7.52 (7H, m),7.61 (2H, d, J=8.8 Hz), 8.19-8.22 (1H, m), 8.54 (1H, s), 10.02 (1H, s);MS m/z (M+H)⁺328.

Anal. for C₂₂H₁₇NO₂.0.1 H₂O:

Calc'd: C, 80.27; H, 5.27; N, 4.25.

Found: C, 80.15; H, 5.05; N, 3.97.

EXAMPLE 2g1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde

Phosphorous oxychloride (3 mL) was added to anhydrous dimethylformamide(6 mL) and the mixture allowed to stir at room temperature for 15 min. Amixture of 1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole (1.03 g,3.1 mmol) in dimethylformamide (10 mL) was added and the mixture heatedto 80° C. and held for 18 hr. The reaction mixture was poured onto iceand adjusted to a pH of 7 by the addition of 2 N NaOH. The mixture wasextracted with ethyl acetate (2×100 mL), the organic layer washed withwater and brine and dried over MgSO₄, and evaporated to provide theproduct as a dark solid. Purification by silica gel chromatography (25%EtOAC-hexanes) provided a white solid (0.84 gr, 76%): mp 166-168-152°C.; ¹H NMR (DMSO-d₆): δ 2.55 (3H, s), 5.30 (2H, s), 7.04 (1H, d, J=6.9Hz), 7.19-7.28 (2H, m), 7.23-7.52 (7H, m), 7.44 (2H, dd, J=6.0 Hz, J=1.4Hz ), 7.59 (1H, dd, , J=11.7 Hz, J=2.5), 8.17 (1H, dd, J=7.0 Hz, J=1.0),10.20 (1H, s) Hz; MS m/z (M+H)⁺360:

Anal. for C₂₃H₁₈FNO₂ (0.1 H₂O):

Calc'd: C, 76.48; H, 5.08; N, 3.88.

Found: C, 76.39; H, 4.81; N, 3.69

EXAMPLE 3a 1-(4-Benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime

A solution of 1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde(0.64 g, 1.9 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol),methanol (25 mL) and pyridine (1 mL) was heated to reflux for 30 min.The mixture was cooled, the methanol removed by evaporation. The productwas extracted into ethyl acetate (2×50 mL) and the organic layer waswashed with water and brine and dried over MgSO₄. The solvent wasevaporated, and the product was purified by silica gel chromatography(25% ethyl acetate-hexanes) to provide a white solid (53%): mp 171-173°C.; ¹H NMR (DMSO-d₆): δ 2.31 (3H, s), 5.20 (2H, s), 6.94-7. J01 (1H, m),7.10-7.14 (2H, m), 7.23 (2H, d, J=8.9 Hz), 7.37-7.47 (5H, m), 7.52 (2H,d, J=7.4 Hz), 8.01-8.03 (1H, m), 8.41 (1H, s), 10.66 (1H, s); MS m/z(M+H)⁺357.

Anal. for C₂₃H₂₀N₂O₂.0.5 H₂O:

Calc'd: C, 75.60; H, 5.79; N, 7.67.

Found: C, 75.12; H, 5.53; N, 7.34.

EXAMPLE 3b5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime

A mixture of5-fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde (0.97g, 2.7 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol), methanol(25 mL) and pyridine (1 mL) was reacted according to the procedure inExample 3a to afford white solid (88%): mp 183-185° C.; ¹H NMR(DMSO-d₆): δ 2.30 (3H, s), 5.20 (2H, s) d, 6.94-6.97 (2H, m), 7.23 (2H,d, J=8.9 Hz), 7.37-7.46 (5H, m), 7.52 (2H, d, J=6.8 Hz), 7.71 (1H, d,J=10.1 Hz), 8.41 (1H, s), 10.72 (1H, s); MS m/z (M+H)⁺375.

Anal. for C₂₃H₁₉FN₂O₂:

Calc'd: C, 73.78; H, 5.11 N, 7.48.

Found: C, 73.55; H, 5.03; N, 7.35.

EXAMPLE 3e(4E)-9-(4-benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one oxime

A mixture of(4E)-9-(4-benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one (1.00 g,2.7 mmol), hydroxylamine hydrochloride (0.56 g, 8.1 mmol), methanol (25mL) and pyridine (1 mL) was reacted according to the procedure inExample 3a to afford a white solid (51%): mp 241-243° C.; ¹H NMR(DMSO-d₆): δ 1.83-1.92 (2H, m), 2.65 (2H, t, J=5.9 Hz), 2.72 (2H, t,J=6.0 Hz), 5.20 (2H, s), 7.09-7.14 (3H, m), 7.22 (2H, d, J=8.8 Hz),7.36-7.46 (5H, m), 7.51 (2H, d, J=6.9 Hz), 8.00-8.02 (1H, m), 10.46 (1H,s); MS m/z (M−H)⁻379.

Anal. for C₂₅H₂₄N₂O₂:

Calc'd: C, 78.51; H, 5.80 N, 7.32.

Found: C, 78.24; H, 5.52; N, 7.26.

EXAMPLE 3f 1-(4-Benzyloxyphenyl)-H-indole-3-carbaldehyde oxime

A mixture of 1-(4-benzyloxyphenyl)-H-indole-3-carbaldehyde (0.50 g, 1.5mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol), methanol (25 mL)and pyridine (1 mL) was reacted according to the procedure in Example 3ato afford a yellow solid (61%): mp 152-154° C.; ¹H NMR (DMSO-d₆): δ 5.20(2H, s), 7.19-7.53 (12H, m), 7.86 (1H, s), 8.10 (1H, d, J=7.5 Hz), 10.75(1H, s

MS m/z (M+H)⁺383. Anal. for C₂₂H₁₈N₂O₂:

Calc'd: C, 77.17; H, 5.30; N, 8.18.

Found: C, 77.49; H, 5.44; N, 7.85.

EXAMPLE 3g1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde oxime

To a stirred solution of1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde (0.81g, 2.3 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol) andmethanol (25 mL) was added pyridine (1 mL). The reaction mixture washeated to reflux and held for 30 min. The mixture was cooled, themethanol removed by evaporation and the residue taken up in ethylacetate/water. The product was extracted into ethyl acetate (2×50 mL),the ethyl acetate washed with water and brine and dried over MgSO₄. Thesolvent was evaporated, and the crude product was purified by silica gelchromatography (25% ethyl acetate-hexanes) to provide a white solid (0.7gr, 83%): mp 167-168° C.; ¹H NMR (DMSO-d₆): δ 2.32 (3H, s), 5.29 (2H,s), 6.99-7.01 (1H, m), 7.10-7.17 (2H, m), 7.25-7.28 (1H, m), 7.37-7.42(1H, m), 7.43-7.49 (3H, m), 7.49-7.54 (2H, m), 8.01-8.04 (1H, m), 8.41(1H, s), 10.67 (1H, s); MS m/z (M+H)⁺375:

Anal. for C₂₃H₁₉FN₂O₂:

Calc'd: C, 73.78; H, 5.11; N, 7.48.

Found: C, 73.53; H, 5.06; N, 7.32

EXAMPLE 4 1-(4-Hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime

To a stirred solution of dichloromethane (20 mL), Pd(OAc)₂ (50 mg, 0.2mmol), and triethylsilane (2 mL) was added triethylamine (1 mL), and themixture was stirred at room temperature for 15 min. To the mixture wasadded 1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime(0.36 g, 1 mmol) in dichloromethane (10 mL), and stirred for 90 min.Ethyl acetate (25 mL) and ammonium chloride (25 mL of 10% solution) wereadded and the product extracted into ethyl acetate (2×25 mL). Thecombined organic layers were washed with water (3×25 mL) and brine (25mL) and dried over MgSO₄. After removing the solvent, a liquid wasobtained, which was dissolved in anhydrous tetrahydrofuran (10 mL). Tothis mixture was added tetrabutyl ammonium fluoride (3 mL). Afterstirring for 30 min, the mixture was extracted with ethyl acetate andwater. The organic layer was washed with water and brine and dried overMgSO₄. After evaporation of the solvent afforded the crude solid whichwas purified by silica gel chromatography (25% ethyl acetate-hexanes) togive a pale yellow solid (83%): mp 171-173° C.; ¹H NMR (DMSO-d₆): δ 2.29(3H, s), 6.95 (4H, d, J=7.7 Hz), 7.09-7.13 (2H, m), 7.22 (2H, d, J=8.7Hz), 7.98-8.01 (1H, m), 8.40 (1H, s), 9.89 (1H, s), 10.63 (1H, s); MSm/z (M+H)⁺267.

Anal. for C₁₆H₁₄N₂O₂:

Calc'd: C, 72.17; H, 5.30; N, 10.52.

Found: C, 71.78; H, 5.23; N, 10.37.

EXAMPLE 5 5-Fluoro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehydeoxime

5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime(0.49 g, 1.3 mmol) was reacted according to the procedure used inexample 4 to afford a white solid (67%): mp 162-163° C.; ¹H NMR(DMSO-d₆): δ 2.29 (3H, s), 6.94-6.97 (4H, m) d, 7.23 (2H, d, J=8.6 Hz),7.70 (2H, d, J=10.1 Hz), 8.39 (1H, s), 9.92 (1H, s), 10.70 (1H, s); MSm/z (M+H)⁺285.

Anal. for C₁₆H₁₃FN₂O₂:

Calc'd: C, 67.60; H, 4.61; N, 9.85.

Found: C, 67.44; H, 4.67; N, 9.79.

EXAMPLE 6 5-Chloro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehydeoxime

A mixture of5-chloro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde (0.63 g,2.2 mmol), hydroxylamine hydrochloride (0.30 g, 4.3 mmol), methanol (25mL) and pyridine (1 mL) was reacted according to the procedure used inExample 4 to afford a white solid (68%): mp 212-213° C.; ¹H NMR(DMSO-d₆): δ 2.29 (3H, s), 6.96 (3H, d, J=8.7 Hz), 7.12 (1H, dd, J=2.2Hz, J=8.7 Hz), 7.24 (2H, d, J=8.7 Hz), 8.03 (1H, d, J=2.1 Hz), 8.40 (1H,s), 9.93 (1H, s), 10.75 (1H, s); MS m/Z (M+H)⁺301/303 (1 Cl).

Anal. for C₁₆H₁₃CIN₂O₂:

Calc'd: C, 63.90; H, 4.36 N, 9.31.

Found: C, 63.63; H, 4.12; N, 9.17.

EXAMPLE 7 5-Bromo-1-(4-hydroxyphenyl)-2-methyl-1H-indole -3-carbaldehydeoxime

A mixture of5-bromo-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde (0.16 g,0.5 mmol), hydroxylamine hydrochloride (0.10 g, 1.4 mmol), methanol (10mL) and pyridine (0.2 mL) was reacted according to the procedure used inExample 4 to afford a white solid (66%): mp 209-211° C.; ¹H NMR(DMSO-d₆): δ 2.29 (3H, s), 6.92-6.97 (3H, m), 7.20-7.25 (3H, m), 8.19(1H, d, J=1.9 Hz), 8.39 (1H, s), 9.94 (1H, br s), 10.76 (1H, s); MS m/z(M+H)⁺345/347 (1 Br).

Anal. for C₁₆H₁₃BrN₂O₂.0.5 H₂O;

Calc'd: C, 54.26; H, 3.98 N, 7.91.

Found: C, 54.26; H, 3.68; N, 7.65.

EXAMPLE 8 (4E)-9-(4-Hydroxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-oneoxime

(4E)-9-(4-Benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one oxime(0.23 g, 0.6 mmol) was reacted according to the procedure used inExample 4 to afford a tan solid (40%): mp 227-229° C.; ¹H NMR (DMSO-d₆):δ 1.87-1.91 (2H, m), 2.63 (2H, t, J=5.9 Hz), 2.71 (2H, t, J=6.1 Hz),6.95 (2H, d, J=8.6 Hz), 7.09-7.13 (3H, m), 7.26 (2H, d, J=8.5 Hz),7.98-8.00 (1H, m), 9.85 (1H, s), 10.43 (1H, s); MS m/z (M+H)⁺293.

Anal. for C₁₈H₁₆N₂O₂:

Calc'd: C, 73.95; H, 5.52; N, 9.58.

Found: C, 73.61; H, 5.39; N, 9.42.

EXAMPLE 9 1-(4-Hydroxyphenyl)-1H-indole-3-carbaldehyde oxime

1-(4-Benzyloxyphenyl)-H-indole-3-carbaldehyde oxime (0.31 g, 0.9 mmol)was reacted according to the procedure used in Example 4 to afford ayellow solid (18%): mp 95-97° C.; ¹H NMR (DMSO-d₆): δ 6.94-6.98 (2H, m),7.19-7.29 (2H, m), 7.36-7.45 (4H, m), 8.31 (1H, s), 9.80-9.83 (1H, m),10.72 (1H, s), 11.44 (1H, s): MS m/z (M+H)⁺253.

Anal. for C₁₅H₁₂N₂O₂:

Calc'd: C, 71.42; H, 4.79; N, 11.10.

Found: C, 71.85; H, 5.36; N, 9.70.

EXAMPLE 101-(3-Fluoro-4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime

To a stirred solution of dichloromethane (10 mL), Pd(OAc)₂ (50 mg, 0.2mmol), and triethylsilane (3 mL) was added triethylamine (1.5 mL), andthe mixture was stirred at ambient temperature for 15 min. To themixture was added1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde oxime(0.52 g, 1.4 mmol) in dichloromethane (15 mL), and stirring wascontinued for 4 hr. Ethyl acetate (25 mL) and ammonium chloride (25 mLof 10% solution) were added and the product extracted into ethyl acetate(2×25 mL). The combined organics were washed with water (3×25 mL) andbrine (25 mL) and dried over MgSO₄. After removing the solvent, a liquidwas obtained, which was dissolved in anhydrous tetrahydrofuran (10 mL).To this stirring mixture was added tetrabutyl ammonium fluoride (3 mL).After stirring for 30 min., the mixture was taken up in ethylacetate/water and shaken and the layers separated. The organic portionwas washed with water and brine and dried over MgSO₄. After evaporationof the solvent, the crude solid was purified by silica gelchromatography (25% ethyl acetate-hexanes) to give a pale yellow solid(0.22 g, 56%): mp 182-184° C.; ¹H NMR (DMSO-d₆): δ 2.31 (3H, s),6.98-7.01 (1H, m), 7.07-7.17 (4H, m), 7.35 (1H, dd, J=11.7 Hz, J=2.4Hz), 7.99-8.03 (1H, m), 8.40 (1H, s), 10.35 (1H, br s), 10.64 (1H, s);MS m/z (M+H)⁺285:

Anal. for C₁₆H₁₃FN₂O₂ (0.3 H₂O):

Calc'd: C, 66.34; H, 4.57 N, 9.30.

Found: C, 66.38; H, 4.61; N, 9.85.

EXAMPLE 11 Selectivity for ERα or ERβ Receptors

Representative examples of the invention were evaluated for theirability to compete with 17β-estradiol for both ERα and ERβ. This testprocedure provides the methodology for one to determine whether aparticular commpound binds to the estrogen receptor (and is therefore“estrogenic”) and whether there is selectivity for ERα or ERβ. Thevalues are shown in the Table infra and are reported as IC₅₀s.17β-estradiol is included as a standard reference for comparison. Theprocedure used is briefly described below. A crude lysate of E. coliexpressing the estrogen receptor ligand binding domains (D, E, & F) ofhuman ERα or ERβ was prepared. Both receptors and compounds were dilutedin 1× Dulbecco's PBS (DPBS) supplemented with 1 mM EDTA. Using a highbinding masked microtiter plate, 100 uL of receptor (1 uG/well) wascombined with 2 nM [³H]-17β-estradiol and various concentrations ofcompound. After between 5 and 15 hours at room temperature, the plateswere washed with DPBS/1 mM EDTA and bound radioactivity determined byliquid scintillation counting. The IC₅₀ is defined as the concentrationof compound that decreases total 17β-estradiol binding by 50%. Theresults obtained are described in the table below. TABLE 11-(4′-Hydroxy-phenyl)-1H-indole-3-carbaldehyde Oxime Derivatives

Ex. R1 R2 R3 R4 ER_(β)(nM) ER_(α)(nM) 4 Me H H H 12 270 5 Me H F H 6.291 6 Me H Cl H 29 145 7 Me H Br H 93.5 235 8 —CH₂CH₂CH₂— H H 10 153 9 HH H H 202 1905 10 Me H H F 8 298

The results obtained in the standard pharmacologic test proceduredemonstrate that the compounds of this invention are estrogeniccompounds, some with strong preferential affinity for the ERβ receptor.The compounds of this invention range from having high preferentialaffinity for ERβ over ERα to almost equal affinity for both receptors.Thus, compounds of this invention will span a range of activity based,at least partially, on their receptor affinity selectivity profiles.Additionally, since each novel receptor ligand complex is unique andthus its interaction with various coregulatory proteins is unique,compounds of this invention will display different modulatory behaviordepending on the cellular context they are in. For example, in somecell-types, it is possible for a compound to behave as an estrogenagonist while in other tissues, an antagonist. Compounds with suchactivity have sometimes been referred to as SERMs (Selective EstrogenReceptor Modulators). Unlike many estrogens, however, many of the SERMsdo not cause increases in uterine wet weight. These compounds areantiestrogenic in the uterus and can completely antagonize the trophiceffects of estrogen agonists in uterine tissue. These compounds,however, act as estrogen agonists in the bone, cardiovascular, andcentral nervous systems. Due to this tissue selective nature of thesecompounds, they are useful in treating or preventing in a mammal diseasestates or syndromes which are caused or associated with an estrogendeficiency (in certain tissues such as bone or cardiovascular) or anexcess of estrogen (in the uterus or mammary glands).

Even beyond such cell-specific modulation, compounds of this inventionalso have the potential to behave as agonists on one receptor type whilebehaving as antagonists on the other. For example, it has beendemonstrated that compounds can be an antagonist on ERβ while being anagonist on ERα (Meyers, Marvin J.; Sun, Jun; Carlson, Kathryn E.;Katzenellenbogen, Benita S.; Katzenellenbogen, John A. J. Med. Chem.(1999), 42(13), 2456-2468). Such ERSAA (Estrogen Receptor SelectiveAgonist Antagonist) activity provides for pharmacologically distinctestrogenic activity within this series of compounds.

Standard pharmacological test procedures are readily available todetermine the activity profile of a given test compound. The followingbriefly summarizes several representative test procedures. Standardpharmacological test procedures for SERMs are also provided in U.S. Pat.Nos. 4,418,068 and 5,998,402.

EXAMPLE 12 Rat Uterotrophic/Antiuterotrophic Test Procedure

The estrogenic and antiestrogenic properties of the compounds can bedetermined in an immature rat uterotrophic assay (4 day) that (asdescribed previously by L. J. Black and R. L. Goode, Life Sciences, 26,1453 (1980). Immature Sprague-Dawley rats (female, 18 days old) weretested in groups of six. The animals are treated by daily ip injectionwith 10 uG compound, 100 uG compound, (100 uG compound+1 uG17β-estradiol) to check antiestrogenicity, and 1 uG 17β-estradiol, with50% DMSO/50% saline as the injection vehicle. On day 4 the animals aresacrificed by CO₂ asphyxiation and their uteri removed and stripped ofexcess lipid, any fluid removed and the wet weight determined. A smallsection of one horn is submitted for histology and the remainder used toisolate total RNA in order to evaluate complement component 3 geneexpression.

EXAMPLE 13 6-Week Ovariectomized Rat Test Procedure—Bone andCardioprotection

Female Sprague Dawley CD rats, ovx or sham ovx, are obtained 1 day aftersurgery from Taconic Farm (weight range 240-275 g). They are housed 3 or4 rats/cage in a room on a 12/12 (light/dark) schedule and provided withfood (Purina 5K96C rat chow) and water ad libitum. Treatment for allstudies begin 1 day after the animals arrival and dosed 7 days per weekas indicated for 6 weeks. A group of age matched sham operated rats notreceiving any treatment serve as an intact, estrogen replete controlgroup for each study.

All treatments are prepared in 1% tween 80 in normal saline at definedconcentrations so that the treatment volume is 0.1 mL/100 g body weight.17β-estradiol is dissolved in corn oil (20 μg/mL) and deliveredsubcutaneously, 0.1 mL/rat. All dosages are adjusted at three weekintervals according to group mean body weight measurements.

Five weeks after the initiation of treatment and one week prior to thetermination of the study, each rat is evaluated for bone mineral density(BMD). The total and trabecular density of the proximal tibia areevaluated in anesthetized rats using an XCT-960M (pQCT; StratecMedizintechnik, Pforzheim, Germany). The measurements are performed asfollows: Fifteen minutes prior to scanning, each rat is anesthetizedwith an intraperitoneal injection of 45 mg/kg ketamine, 8.5 mg/kgxylazine, and 1.5 mg/kg acepromazine.

The right hind limb is passed through a polycarbonate tube with adiameter of 25 mm and taped to an acrylic frame with the ankle joint ata 90° angle and the knee joint at 180°. The polycarbonate tube isaffixed to a sliding platform that maintains it perpendicular to theaperture of the pQCT. The platform is adjusted so that the distal end ofthe femur and the proximal end of the tibia would be in the scanningfield. A two dimensional scout view is run for a length of 10 mm and aline resolution of 0.2 mm. After the scout view is displayed on themonitor, the proximal end of the tibia is located. The pQCT scan isinitiated 3.4 mm distal from this point. The pQCT scan is 1 mm thick,has a voxel (three dimensional pixel) size of 0.140 mm, and consists of145 projections through the slice.

After the pQCT scan is completed, the image is displayed on the monitor.A region of interest including the tibia but excluding the fibula isoutlined. The soft tissue is automatically removed using an iterativealgorithm. The density of the remaining bone (total density) is reportedin mg/cm³. The outer 55% of the bone is peeled away in a concentricspiral. The density of the remaining bone (Trabecular density) isreported in mg/cm³. One week after BMD evaluation the rats areeuthanized by carbon dioxide suffocation and blood collected forcholesterol determination. The uteri are removed and the weights taken.Total cholesterol is determined using a Boehringer-Mannheim Hitachi 911clinical analyzer using the Cholesterol/HP kit. Statistics were comparedusing one-way analysis of variance with Dunnet's test.

EXAMPLE 14 MCF-7/ERE Antiproliferative Test Procedure

Stock solutions of test compounds (usually 0.1 M) are prepared in DMSOand then diluted 10 to 100-fold with DMSO to make working solutions of 1or 10 mM. The DMSO stocks are stored at either 4° C. (0.1 M) or −20° C.(<0.1M). MCF-7 cells are passaged twice a week with growth medium[D-MEM/F-12 medium containing 10% (v/v) heat-inactivated fetal bovineserum, 1% (v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1]. The cellsare maintained in vented flasks at 37° C. inside a 5% CO₂/95% humidifiedair incubator. One day prior to treatment, the cells are plated withgrowth medium at 25,000/well into 96 well plates and incubated at 37° C.overnight.

The cells are infected for 2 hr at 37° C. with 50 μl/well of a 1:10dilution of adenovirus 5-ERE-tk-luciferase in experimental medium[phenol red-free D-MEM/F-12 medium containing 10% (v/v) heat-inactivedcharcoal-stripped fetal bovine serum, 1% (v/v) Penicillin-Streptomycin,2 mM glutaMax-1, 1 mM sodium pyruvate]. The wells are then washed oncewith 150 μλ of experimental medium. Finally, the cells are treated for24 hr at 37° C. in replicates of 8 wells/treatment with 150 μλ/well ofvehicle (≦0.1% v/v DMSO) or compound that is diluted ≧1000-fold intoexperimental medium.

Initial screening of test compounds is done at a single dose of 1 μMthat is tested alone (agonist mode) or in combination with 0.1 nM17β-estradiol (EC₈₀; antagonist mode). Each 96 well plate also includesa vehicle control group (0.1% v/v DMSO) and an agonist control group(either 0.1 or 1 nM 17β-estradiol). Dose-response experiments areperformed in either the agonist and/or antagonist modes on activecompounds in log increases from 10⁻¹⁴ to 10⁻⁵ M. From thesedose-response curves, EC₅₀ and IC₅₀ values, respectively, are generated.The final well in each treatment group contains 5 μl of 3×10⁻⁵ MICI-182,780 (10⁻⁶ M final concentration) as an ER antagonist control.

After treatment, the cells are lysed on a shaker for 15 min with 25μl/well of 1X cell culture lysis reagent (Promega Corporation). The celllysates (20 μl ) are transferred to a 96 well luminometer plate, andluciferase activity is measured in a MicroLumat LB 96 P luminometer (EG& G Berthold) using 100 μl/well of luciferase substrate (PromegaCorporation). Prior to the injection of substrate, a 1 second backgroundmeasurement is made for each well. Following the injection of substrate,luciferase activity is measured for 10 seconds after a 1 second delay.The data are transferred from the luminometer to a Macintosh personalcomputer and analyzed using the JMP software (SAS Institute); thisprogram subtracts the background reading from the luciferase measurementfor each well and then determines the mean and standard deviation ofeach treatment.

The luciferase data are transformed by logarithms, and the HuberM-estimator is used to down-weight the outlying transformedobservations. The JMP software is used to analyze the transformed andweighted data for one-way ANOVA (Dunnett's test). The compoundtreatments are compared to the vehicle control results in the agonistmode, or the positive agonist control results (0.1 nM 17β-estradiol) inthe antagonist mode. For the initial single dose experiment, if thecompound treatment results are significantly different from theappropriate control (p<0.05), then the results are reported as thepercent relative to the 17β-estradiol control [i.e., ((compound−vehiclecontrol)/(17β-estradiol control−vehicle control))×100]. The JMP softwareis also used to determine the EC₅₀ and/or IC₅₀ values from thenon-linear dose-response curves.

EXAMPLE 15 Inhibition of LDL Oxidation—Antioxidant Activity

Porcine aortas are obtained from an abattoir, washed, transported inchilled PBS, and aortic endothelial cells are harvested. To harvest thecells, the intercostal vessels of the aorta are tied off and one end ofthe aorta clamped. Fresh, sterile filtered, 0.2% collagenase (Sigma TypeI) is placed in the vessel and the other end of the vessel then clampedto form a closed system. The aorta is incubated at 37° C. for 15-20minutes, after which the collagenase solution is collected andcentrifuged for 5 minutes at 2000×g. Each pellet is suspended in 7 mL ofendothelial cell culture medium consisting of phenol red free DMEM/Ham'sF12 media supplemented with charcoal stripped FBS (5%), NuSerum (5%),L-glutamine (4 mM), penicillin-streptomycin (100 U/ml, 100 μg/ml) andgentimicin (75 μg/ml), seeded in 100 mm petri dish and incubated at 37°C. in 5% CO₂. After 20 minutes, the cells are rinsed with PBS and freshmedium added, this was repeated again at 24 hours. The cells areconfluent after approximately 1 week. The endothelial cells areroutinely fed twice a week and, when confluent, trypsinized and seededat a 1:7 ratio. Cell mediated oxidation of 12.5 μg/mL LDL is allowed toproceed in the presence of the compound to be evaluated (5 μM) for 4hours at 37° C. Results are expressed as the percent inhibition of theoxidative process as measured by the TBARS (thiobarbituric acid reactivesubstances) method for analysis of free aldehydes (Yagi K., Biochem Med15:212-216 (1976)).

EXAMPLE 16 D12Hypothalmic Cell Test Procedure

D12 rat hypothalamic cells are subcloned from the RCF17 parental cellline and stored frozen. They are routinely grown in DMEM:F12 (1:1),glutaMAX-1 (2 mM), penicillin (100 U/ml)-streptomycin (100 mg/ml), plus10% fetal bovine serum (FBS). The cells are plated in phenol red-freemedium (DMEM:F12, glutaMAX, penicillin-streptomycin) containing 2-10%charcoal stripped FBS at a subconfluent density (1-4×10 6 cells/150 mmdish). The cells are refed 24 h later with medium containing 2% strippedserum. To test for agonist activity, cells are treated with 10 nM17b-estradiol or various doses of test compound (1 mM or a range from 1pM to 1 mM). To test for antagonist activity the cells are treated with0.1 nM 17β-estradiol in the absence or presence of varying doses (100 pMto 1 mM) of test compound. Control dishes are also treated with DMSO asa negative control. Forty-eight hours after hormone addition, the cellsare lysed and binding test procedure performed.

For each binding test procedure 100-150 mg protein is incubated with 10nM ³H-R5020+100-fold excess R5020 in a 150 ml volume. Triplicatereactions (three with R5020, three without R5020) are prepared in a 96well plate. The protein extract is added first followed by ³H-R5020 or³H-R5020+100× unlabeled R5020. The reaction is performed for 1-2 hr atroom temperature . The reaction is stopped by the addition of 100 mlcold 5% charcoal (Norit SX-4), 0.5% dextran 69 K (Pharmacia) in TE pH7.4 . After 5 min at room temperature, the bound and unbound ligand areseparated by centrifugation (5 min, 1000 RCF, 4° C.). The supernatantsolution (˜150 ml) is removed and transferred to a scintillation vial.Following the addition of scintillation fluid (Beckman Ready Protein+),the samples are counted for 1 min in a scintillation counter.

EXAMPLE 17 Progesterone Receptor in the CNS Preoptic Area

Sixty (60) day old female Sprague-Dawley rats are ovariectomized. Theanimals are housed in an animal care facility with a 12-h light, 12-hdark photoperiod and free access to tap water and rodent chow.

Ovariectomized animals are randomly divided into groups that areinjected with vehicle (50% DMSO, 40% PBS, 10% ethanol vehicle),17β-estradiol (200 ng/kg) or the compound to be tested. Additionalanimals are injected with the test compound 1 hr prior to injection of17β-estradiol to evaluate the antagonistic properties of this compound.Six hrs after s.c. injection, animals are euthanized with a lethal doseof CO₂ and their brains collected and frozen.

Tissue collected from animals is cut on a cryostat at −16° C. andcollected on Silane-coated microscope slides. The section-mounted slidesare then dried on a slide warmer maintained at 42° C. and stored indesiccated slide boxes at −80° C. Prior to processing, the desiccatedslide boxes are slowly warmed to room temperature (−20° C. for 12-16hrs; 4° C. for 2 hrs; room temperature for 1 hr) to eliminatecondensation formation on slides and thus minimize tissue and RNAdegradation. The dry slides are loaded into metal racks, postfixed in 4%paraformaldehyde (pH 9.0) for 5 min and processed as previouslydescribed.

A plasmid containing a 815 bp fragment of the rat PR cDNA 9 (ligandbinding domain) is linearized and used to generate a S 35-UTP labeledprobe that is complimentary to a portion of the rat PR mRNA. Processedsection-mounted slides are hybridized with 20 ml of hybridization mixcontaining the riboprobe (4-6×106 DPM/slide) and 50% formamide andincubated overnight in a 55° C. humidified chamber. In the morning, theslides are placed in metal racks that are immersed in 2×SSC (0.15M NaCl,0.015M sodium citrate; pH 7.0)/10 mM DTT. The racks are all transferredto a large container and washed in 2×SSC/10 mM DTT for 15 min at RT withgentle agitation. Slides are then washed in RNase buffer at 37° C. for30 min, treated with RNase A (2 mg/ml) for 30 min at 37° C., and washedfor 15 min in room temperature 1×SSC. Subsequently, the slides arewashed (2×30 min) in 65° C. in 0.1×SSC to remove nonspecific label,rinsed in room temperature 0.1×SSC for 15 min and dehydrated with agraded series of alcohol: ammonium acetate (70%, 95%, and 100%). Airdried slides are opposed to x-ray film for 3 days and thenphotographically processed. The slides from all animals are hybridized,washed, exposed and photographically processed together to eliminatedifferences due to interassay variation in conditions.

EXAMPLE 18 Rat Hot Flush—CNS Effects

Ovariectomized-female, 60 day-old Sprague-Dawley rats are obtainedfollowing surgery. The surgeries are done a minimum of 8 days prior tothe first treatment. The animals are housed individually under 12 hlight/dark cycle and given standard rat chow and water ad libitum.

Two control groups are included in every study. Doses are prepared basedon mg/kg mean group body weight in either 10% DMSO in sesame oil (scstudies) or in 1.0% tween 80 in saline (po studies). Animals areadministered test compounds at doses ranging from 0.01 to 10 mg/kg meangroup body weight. Vehicle and ethinyl estradiol (EE) controls (0.1mg/kg, sc or 0.3 mg/kg, po) control groups are included in each test.When the compounds are tested for their antagonist activity, EE iscoadministered at 0.1 or 0.3 mg/kg for sc or po studies, respectively.The test compounds are administered up to the day tail skin temperatureis measured.

After the acclimation period of four days, the animals are treated oncedaily with the compound(s) of interest. There are 10 animals/treatmentgroup. Administration of the compound is either by sc injection of 0.1ml in the nape of the neck or po in a volume of 0.5 ml. On the 3rd dayof treatment, a morphine pellet (75 mg morphine sulfate) is implantedsubcutaneously. On the 5th day of treatment, one or two additionalmorphine pellets are implanted. On the eighth day, approximately half ofthe animals are injected with Ketamine (80 mg/kg, intramuscularly) and athermocouple, connected with to a MacLab Data Acquisition System (APIInsturments, Milford, Mass) is taped on the tail approximately one inchfrom the root of the tail. This system allowed the continuousmeasurement of tail skin temperature. Baseline temperature is measuredfor 15 min, then naloxone (1.0 mg/kg) is given sc (0.2 ml) to block theeffect of morphine and tail skin temperature is measured for one hourthereafter. On the ninth day, the remaining of the animals are set upand analyzed similarly.

EXAMPLE 19 Vasomotor Function in Isolated Rat Aortic Rings

Sprage-Dawley rats (240-260 grams) are divided into 4 groups:

-   1. Normal non-ovariectomized (intact)-   2. Ovariectomized (ovex) vehicle treated-   3. Ovariectomized 17β-estradiol treated (1 mg/kg/day)-   4. Ovariectomized animals treated with test compound (i.e., 1    mg/kg/day)

Animals are ovariectomized approximately 3 weeks prior to treatment.Each animal receives 1 mg/kg/day of either 17β-estradiol sulfate or testcompound suspended in distilled, deionized water with 1% tween-80 bygastric gavage. Vehicle treated animals received an appropriate volumeof the vehicle used in the drug treated groups.

Animals are euthanized by CO₂ inhalation and exsanguination. Theirthoracic aortas are rapidly removed and placed in 37° C. physiologicalsolution with the following composition (mM): NaCl (54.7), KCl (5.0),NaHCO₃ (25.0), MgCl₂ 2H₂O (2.5), D-glucose (11.8) and CaCl₂ (0.2) gassedwith CO₂—O₂, 95%/5% for a final pH of 7.4. The advantitia is removedfrom the outer surface and the vessel is cut into 2-3 mm wide rings.Rings are suspended in at 10 mL tissue bath with one end attached to thebottom of the bath and the other to a force transducer. A restingtension of 1 gram is placed on the rings. Rings are equilibrated for 1hour, signals are acquired and analyzed.

After equilibration, the rings are exposed to increasing concentrationsof phenylephrine (10⁻⁸ to 10⁻⁴ M) and the tension recorded. Baths arethen rinsed 3 times with fresh buffer. After washout, 200 mM L-NAME isadded to the tissue bath and equilibrated for 30 minutes. Thephenylephrine concentration response curve is then repeated.

EXAMPLE 20 Eight Arm Radial Arm Maze—Cognition Enhancement

Male Sprague-Dawley, CD rats (Charles River, Kingston, N.Y.) weighing200-250 g on arrival are used. For one week, the rats are housed, sixper cage, with standard laboratory chow and water available ad libitum.Housing is in a colony room maintained at 22° C. and had a 12 hourlight/dark cycle with lights on at 6:00 AM. Following habituation to thefacility, animals are individually housed and maintained at 85% offree-feeding weight. Once stable weights are attained, the rats areacclimated to the 8-arm radial maze.

The structure of the maze is an adaptation from that of Peele and Baron(Pharmacology, Biochemistry, and Behavior, 29:143-150, 1988). The mazeis elevated to a height of 75.5 cm and composed of a circular areasurrounded by 8 arms radiating away from the center, equidistant fromone another. Each arm is 58 cm long×13 cm high. A clear plexiglasscylinder is lowered to enclose the animal in the center portion of themaze prior to the start of each session. Each arm of the maze isequipped with 3 sets of photocells interfaced to a data acquisitionunit, which in turn is interfaced to a computer. The photocells are usedto track the movement of the rat in the maze. Pellet feeders locatedabove food cups at the end of each arm, dispensed two 45 mg chocolatepellets when the outer photocell of the arm is activated for the firsttime in a given session. The maze is located in a testing room withblack and white geometric posters on each wall to serve as visual cues.During all training and testing procedures, white noise is audible (˜70db).

The training procedure consists of five phases, each with daily sessionslasting 5 or 10 minutes. A 10 second delay is imposed between the timethe rat is placed in the center portion of the maze and when thecylinder is raised to begin the session. During Phase 1, food-restrictedpairs of rats are placed on the maze for 10 minutes with 45 mg chocolatefood pellets scattered throughout the 8 arms of the maze. During PhaseII, each rat is placed individually on the maze for a 10 minute period,with pellets scattered from the middle photocell to the food cup of eacharm. During Phase III, each rat is placed on the maze for a 10 minuteperiod, with food pellets located only in and around the food cups ineach arm. In Phase IV, each rat is allowed 10 minutes to collect twopellets from each arm. Re-entry into an arm is considered an error. Ratsare trained daily in this manner until they achieved criterionperformance with less than or equal to 2 total errors on threeconsecutive days of training. Total habituation and training time isapproximately 3 weeks.

Test compound is prepared in phosphate buffered saline and administeredin a volume of 1 ml/kg. Scopolamine HBr (0.3 mg/kg s.c.) served as theimpairing agent, producing an increase in error rate (loss of memory).Test compound is given intraperitoneally simultaneously withscopolamine, 30 minutes prior to the first maze exposure on any giventest day.

To assess the test compound, an 8×8 balanced latin square for repeatedmeasures is designed, in order to achieve a high experimental efficiencywith the least amount of animals. Eight experimental sessions, two perweek, are conducted with the 8 treatments (vehicle, scopolamine, 3 dosesof test compound in combination with scopolamine) randomized within eachsession. Each treatment followed every other treatment the same numberof times. Therefore, the residual effect of every treatment could beestimated and removed from the direct treatment effect. Following ANOVA,multiple comparisons are performed using Dunnett's two-sided test onadjusted means.

Animals that did not make 4 correct choices within 5 minutes during thefirst exposure, or that had not made a total of 8 choices by the end ofthe 2nd exposure, are considered to have “timed-out” for that session.Any animal that “timed-out” following administration of more than onedose of the test compound is excluded from the analysis.

EXAMPLE 21 Neuroprotection Inhibition of Time-Dependent Death of Cellsin Primary Cortical Neuron Cultures

Primary cortical neurons were produced from rat brains that were 0-1 dayold using a variation of methods described by Monyer et al. 1989, BrainResearch 483:347-354. Dispersed brain tissue was grown in DMEM/10% PDHS(pregnant donor horse serum) for three days and then treated withcytosine arabinoside (ARC) for two days to remove contaminating glialcells. On day 5, the ARC media was removed and replaced with DMEM/10%PDHS. The neuronal cells were cultured for a further 4-7 days beforeuse.

Control primary neuronal cultures show progressive cell death betweendays 12 and 18 in culture. Twelve cultures were evaluated on days 12 and16 for levels of the enzyme lactate dehydrogenase (LD) after adding testcompound to 6 cultures maintained in DMEM and 10% PDHS on day 9 andmaintaining the remaining cultures as controls. LD was assayed using avariation of the method by Wroblewski et al. 1955, Proc. Soc. Exp. Biol.Med. 90:210-213. LD is a cytosolic enzyme which is commonly used in bothclinical and basic research to determine tissue viability. An increasein media LD is directly related to cell death.

Neuroprotection Against Cytotoxicity Induced by Hypoglycemia

C6 glioma cells obtained from ATCC were plated in RPMI media with FBS ata concentration of 1×10<6 > cells/ml in FALCON 25 cm² tissue cultureflasks. Four hours prior to the onset of hypoglycemia, the maintenancemedia was discarded, monolayers were washed twice in the appropriatemedia and then incubated for four hours at 37° C. in either serum freeor serum free plus test compound. Kreb's Ringer Phosphate buffer wasused to wash the monolayers twice before the addition of appropriateglucose treatment. RPMI medium contains 2 mg glucose/ml; flasks weredivided into groups of 6 each receiving 100% glucose (2 mg/ml), 80%glucose (1.6 mg/ml), 60% glucose (1.2 mg/ml) or 0% glucose (buffer) orsupplemented with test compound. All flasks were incubated for 20 hoursand then evaluated for total, live, and dead cell number utilizingtrypan blue.

Neuroprotection Against Excitotoxic Amino Acids

Five culture dishes containing SK-N-SH neuroblastoma cells were treatedwith test compound and 5 culture dishes were treated with RPMI media.Four hours later, all cell were treated with NMDA (500 mu M) for 5minutes. Total live cells and dead cells were then determined.

Neuroprotection Against Oxygen-Glucose Deprivation

Analysis of pyknotic nuclei to measure apoptosis: Cortical neurons areprepared from E18 rat fetus and plated in 8-well chamber slidesprecoated with poly-D-lysine (10 ng/ml) and serum at a density of100,000 cells/well. Cells are plated in high glucose DMEM containing 10%FCS and kept in the incubator at 37° C. with 10% CO₂/90% air. On thenext day, serum is removed by replacing culture media with high glucoseDMEM containing B27 supplement and cells are kept in the incubatorwithout further media change until the day of experiment. On day 6,slides are divided into two groups; control group and OGD group. Cellsin control group receive DMEM with glucose and custom B27 (withoutantioxidants). Cells in OGD group receive no-glucose DMEM with customB27, which has been degassed under vacuum for 15 min. Cells are flushedwith 90% N₂/10% CO₂ for 10 min in an airtight chamber and incubated at37° C. for 6 hrs. After 6 hrs, both control and OGD cells are subject toreplacement of media containing either vehicle (DMSO) or test compoundin glucose-containing DMEM with custom B27. Cells are returned tonormoxic incubator at 37° C. After 24 hrs, cells are fixed in 4% PFA for10 min at 4° C. and stained with Topro (Fluorescent nuclear bindingdye). Apoptosis is assessed using Laser Scanning Cytometer by measuringpyknotic nuclei.

Measurement of LDH release as an indication of cell death: Corticalneurons are prepared from E18 rat fetus and plated in 48-well cultureplates precoated with poly-D-lysine (10 ng/ml) and serum at a density of150,000 cells/well. Cells are plated in high glucose DMEM containing 10%FCS and kept in the incubator at 37° C. with 10% CO₂/90% air. On thenext day, serum is removed by replacing culture media with high glucoseDMEM containing B27 supplement. On day 6, cells are divided into twogroups; control group and OGD group. Cells in control group receive DMEMwith glucose and custom B27 (without antioxidants). Cells in OGD groupreceive no-glucose DMEM with custom B27, which has been degassed undervacuum for 15 min. Cells are flushed with 90% N₂/10% CO₂ for 10 min inan airtight chamber and incubated at 37° C. for 6 hrs. After 6 hrs, bothcontrol and OGD cells are subject to replacement of media containingeither vehicle (DMSO) or test compound in glucose-containing DMEM withcustom B27. Cells are returned to normoxic incubator at 37° C. After 24hrs, cell death is assessed by measuring cellular release of LDH(lactate dehydrogenase) into the culture medium. For LDH assay, analiquot of 50 μl culture medium is transferred into the 96 well plate.After the addition of 140 μl 0.1M potassium phosphate buffer (pH 7.5)and 100 μl 0.2 mg/ml NADH, the plate is let sit in the dark at roomtemperature for 20 min. The reaction is initiated by the addition of 10μl of sodium pyruvate. The plate is read immediately at 340 nM in aThermomax plate reader (Molecular Devices). The absorbance, an index ofNADH concentration, is recorded every 6 seconds for 5 minutes and theslope indicating the rate of NADH disappearance is used to calculate LDHactivity.LDHActivity(U/ml)=(ΔA/min)(TCF)(20)(0.0833)/(0.78)

-   -   where: 0.0833=proportionality constant    -   0.78=instrument light path length (cm)

EXAMPLE 22 HLA Rat Test Procedure—Crohn's Disease and Inflammatory BowelDisorders

Male HLA-B27 rats are obtained from Taconic and provided unrestrictedaccess to a food (PMI Lab diet 5001) and water. At the start of thestudy, rats are 22-26 weeks old.

Rats are dosed subcutaneously once per day for seven days with one ofthe formulations listed below. There are five rats in each group and thelast dose is administered two hours before euthanasia.

-   -   vehicle (50% DMSO/50% Dulbecco's PBS)    -   17α-ethinyl-17β-estradiol (10 μg/kg)    -   test compound

Stool quality is observed daily and graded according to the followingscale: Diarrhea=3; soft stool=2; normal stool=1. At the end of the testprocedure, serum is collected and stored at −70° C. A section of colonis prepared for histological analysis and an additional segment isanalyzed for myeloperoxidase activity.

The following method is used to measure myeloperoxidase activity. Colontissue is harvested and flash frozen in liquid nitrogen. Arepresentative sample of the entire colon is used to ensure consistencybetween samples. The tissue is stored at −80° C. until use. Next, thetissue is weighed (approximately 500 mg) and homogenized in 1:15 w/v of5 mM H₂KPO₄ (pH 6) washing buffer. The tissue is spun down at 20,000×gin a Sorvall RC 5B centrifuge for 45 minutes at 2-8° C. Supernatant isthen discarded. Tissue is resuspended and homogenized in 2.5 ml (1:5w/v) of 50 mM H₂KPO₄ with 10 mM EDTA and 0.5% Hex Ammonium Bromide tohelp solubilize the intracellular MPO. Tissue is frozen in liquidNitrogen, thawed in a 37° C.-water bath and sonicated for 15 seconds toensure membrane lysis. This procedure is repeated 3 times. Samples arethen kept on ice for 20 minutes and centrifuged at 12,000×g for 15minutes at 2-8° C. The supernatant is analyzed following these steps.

The test mixture is prepared by adding 2.9 ml of 50 mM H₂KPO₄ with 0.167O-Dianisidine/ml with 0.0005% H₂O₂ into a reaction tube. When hydrogenperoxide is degraded, O-Dianisidine is oxidized and absorbs at 460 nm ina concentration dependent manner. The mixture is heated to 25° C. Onehundred (100) μL of the tissue supernatant is added to the reactiontube, incubated for one minute at 25° C., then 1 ml is transferred to adisposable plastic cuvette. OD is measured every 2 minutes reaction timeat 460 nm against a blank containing 2.9 ml of the reaction mixture and100 μl of the 0.5% ammonium bromide solution.

Enzyme activity units are quantified by comparison of absorbence @ 460to a standard curve prepared with purified human MPO 31.1 UnitsNial. TheMPO is reconstituted and serially diluted using 50 mM H₂KPO₄ with 10 mMEDTA and 0.5% Hex Ammonium Bromide to four known concentrations. Sampleabsorbencies are compared against this curve to determine activity.

Histological analysis is performed as follows. Colonic tissue isimmersed in 10% neutral buffered formalin. Each specimen of colon isseparated into four samples for evaluation. The formalin-fixed tissuesare processed in a vacuum infiltration processor for paraffin embedding.The samples are sectioned at 5 μm and then stained with hematoxylin andeosin (H&E) for blinded histologic evaluations using a scale modifiedafter Boughton-Smith. After the scores are completed the samples areunblinded, and data are tabulated and analyzed by ANOVA linear modelingwith multiple mean comparisons.

All patents, publications, and other documents cited herein are herebyincorporated by reference in their entirety.

1. A method of inhibiting osteoarthritis, hypocalcemia, hypercalcemia,Paget's disease, osteomalacia, osteohalisteresis, multiple myeloma orother forms of cancer having deleterious effects on bone tissues in amammal in need thereof, comprising providing to said mammal an effectiveamount of a compound of the formula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.
 2. A method ofinhibiting benign or malignant abnormal tissue growth in a mammal inneed thereof, comprising providing to said mammal an effective amount ofa compound of the formula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.
 3. The method ofclaim 2 wherein the abnormal tissue growth is prostatic hypertrophy,uterine leiomyomas, breast cancer, endometriosis, endometrial cancer,polycystic ovary syndrome, endometrial polyps, benign breast disease,adenomyosis, ovarian cancer, melanoma, prostrate cancer, cancers of thecolon, or CNS cancers.
 4. A method of lowering cholesterol,triglycerides, Lp(a), or LDL levels; or inhibiting hypercholesteremia;hyperlipidemia; cardiovascular disease; atherosclerosis; peripheralvascular disease; restenosis, or vasospasm; or inhibiting vascular walldamage from cellular events leading toward immune mediated vasculardamage in a mammal in need thereof, comprising providing to said mammalan effective amount of a compound of the formula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.
 5. A method ofinhibiting free radical induced disease states in a mammal in needthereof, comprising providing to said mammal an effective amount of acompound of the formula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.
 6. A method ofproviding cognition enhancement or neuroprotection; or treating orinhibiting senile dementias, Alzheimer's disease, cognitive decline, orneurodegenerative disorders in a mammal in need thereof, comprisingproviding to said mammal an effective amount of a compound of theformula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.
 7. A method ofinhibiting inflammatory bowel disease, ulcerative proctitis, Crohn'sdisease, colitis, hot flashes, vaginal or vulvar atrophy, atrophicvaginitis, vaginal dryness, pruritus, dyspareunia, dysuria, frequenturination, urinary incontinence, urinary tract infections, vasomotorsymptoms; male pattern baldness; skin atrophy; acne; type II diabetes;dysfunctional uterine bleeding; or infertility in a mammal in needthereof, comprising providing to said mammal an effective amount of acompound of the formula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.
 8. A method ofinhibiting leukemia, endometrial ablations, chronic renal or hepaticdisease or coagulation diseases or disorders in a mammal in needthereof, comprising providing to said mammal an effective amount of acompound of the formula:

wherein: R₁ is hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R₂is hydrogen, lower alkyl, or phenyl; or R₁ and R₂ together may form a5-7 membered ring; and R₃, and R₄ are each, independently, H, OH,halogen, CN, phenyl, substituted phenyl, lower alkyl or lower alkoxy; ora pharmaceutically acceptable salt or prodrug thereof.